Retrograde migration of pectoral girdle muscle precursors depends on CXCR4/SDF-1 signaling

In vertebrates, muscles of the pectoral girdle connect the forelimbs with the thorax. During development, the myogenic precursor cells migrate from the somites into the limb buds. Whereas most of the myogenic precursors remain in the limb bud to form the forelimb muscles, several cells migrate back toward the trunk to give rise to the superficial pectoral girdle muscles, such as the large pectoral muscle, the latissimus dorsi and the deltoid. Recently, this developing mode has been referred to as the “In–Out” mechanism. The present study focuses on the mechanisms of the “In–Out” migration during formation of the pectoral girdle muscles. Combining in ovo electroporation, tissue slice-cultures and confocal laser scanning microscopy, we visualize live in detail the retrograde migration of myogenic precursors from the forelimb bud into the trunk region by live imaging. Furthermore, we present for the first time evidence for the involvement of the chemokine receptor CXCR4 and its ligand SDF-1 during these processes. After microsurgical implantations of CXCR4 inhibitor beads in the proximal forelimb region of chicken embryos, we demonstrate with the aid of in situ hybridization and live-cell imaging that CXCR4/SDF-1 signaling is crucial for the retrograde migration of pectoral girdle muscle precursors. Moreover, we analyzed the MyoD expression in CXCR4-mutant mouse embryos and observed a considerable decrease in pectoral girdle musculature. We thus demonstrate the importance of the CXCR4/SDF-1 axis for the pectoral girdle muscle formation in avians and mammals.     

Molecular Basis for Broad Neuraminidase Immunity: Conserved Epitopes in Seasonal and Pandemic H1N1 as Well as H5N1 Influenza Viruses

Influenza A viruses, including H1N1 and H5N1 subtypes, pose a serious threat to public health. Neuraminidase (NA)-related immunity contributes to protection against influenza virus infection. Antibodies to the N1 subtype provide protection against homologous and heterologous H1N1 as well as H5N1 virus challenge. Since neither the strain-specific nor conserved epitopes of N1 have been identified, we generated a panel of mouse monoclonal antibodies (MAbs) that exhibit different reactivity spectra with H1N1 and H5N1 viruses and used these MAbs to map N1 antigenic domains. We identified 12 amino acids essential for MAb binding to the NA of a recent seasonal H1N1 virus, A/Brisbane/59/2007. Of these, residues 248, 249, 250, 341, and 343 are recognized by strain-specific group A MAbs, while residues 273, 338, and 339 are within conserved epitope(s), which allows cross-reactive group B MAbs to bind the NAs of seasonal H1N1 and the 1918 and 2009 pandemic (09pdm) H1N1 as well as H5N1 viruses. A single dose of group B MAbs administered prophylactically fully protected mice against lethal challenge with seasonal and 09pdm H1N1 viruses and resulted in significant protection against the highly pathogenic wild-type H5N1 virus. Another three N1 residues (at positions 396, 397, and 456) are essential for binding of cross-reactive group E MAbs, which differ from group B MAbs in that they do not bind 09pdm H1N1 viruses. The identification of conserved N1 epitopes reveals the molecular basis for NA-mediated immunity between H1N1 and H5N1 viruses and demonstrates the potential for developing broadly protective NA-specific antibody treatments for influenza.     

Pseudoinfectious Venezuelan Equine Encephalitis Virus: a New Means of Alphavirus Attenuation

Venezuelan equine encephalitis virus (VEEV) is a reemerging virus that causes a severe and often fatal disease in equids and humans. In spite of a continuous public health threat, to date, no vaccines or antiviral drugs have been developed for human use. Experimental vaccines demonstrate either poor efficiency or severe adverse effects. In this study, we developed a new strategy of alphavirus modification aimed at making these viruses capable of replication and efficient induction of the immune response without causing a progressive infection, which might lead to disease development. To achieve this, we developed a pseudoinfectious virus (PIV) version of VEEV. VEE PIV mimics natural viral infection in that it efficiently replicates its genome, expresses all of the viral structural proteins, and releases viral particles at levels similar to those found in wild-type VEEV-infected cells. However, the mutations introduced into the capsid protein make this protein almost incapable of packaging the PIV genome, and most of the released virions lack genetic material and do not produce a spreading infection. Thus, VEE PIV mimics viral infection in terms of antigen production but is safer due to its inability to incorporate the viral genome into released virions. These genome-free virions are referred to as virus-like particles (VLPs). Importantly, the capsid-specific mutations introduced make the PIV a very strong inducer of the innate immune response and add self-adjuvant characteristics to the designed virus. This unique strategy of virus modification can be applied for vaccine development against other alphaviruses.     

Synthesis of New 4-Aminoquinolines and Evaluation of Their In Vitro Activity against Chloroquine-Sensitive and Chloroquine-Resistant Plasmodium falciparum

The efficacy of chloroquine, once the drug of choice in the fight against Plasmodium falciparum, is now severely limited due to widespread resistance. Amodiaquine is one of the most potent antimalarial 4-aminoquinolines known and remains effective against chloroquine-resistant parasites, but toxicity issues linked to a quinone-imine metabolite limit its clinical use. In search of new compounds able to retain the antimalarial activity of amodiaquine while circumventing quinone-imine metabolite toxicity, we have synthesized five 4-aminoquinolines that feature rings lacking hydroxyl groups in the side chain of the molecules and are thus incapable of generating toxic quinone-imines. The new compounds displayed high in vitro potency (low nanomolar IC₅₀), markedly superior to chloroquine and comparable to amodiaquine, against chloroquine-sensitive and chloroquine-resistant strains of P. falciparum, accompanied by low toxicity to L6 rat fibroblasts and MRC5 human lung cells, and metabolic stability comparable or higher than that of amodiaquine. Computational studies indicate a unique mode of binding of compound 4 to heme through the HOMO located on a biphenyl moeity, which may partly explain the high antiplasmodial activity observed for this compound.     

Vaccination campaigns in postsocialist Ukraine: health care providers navigating uncertainty

Vaccination anxieties grew into a public health issue during the 2008 failed measles and rubella immunization campaign in Ukraine. Here I explore how health care providers bend official immunization policies as they navigate media scares about vaccines, parents' anxieties, public health officials' insistence on the need for vaccination, and their own sense of expertise and authority. New hierarchies are currently being renegotiated, and I follow health care providers as they attempt to parcel out their new position in the Ukrainian society and beyond. Public health control is reframed in a postsocialist context as a condition of acceptance into the European community as a sanitary democracy, and a contestation point between citizens and state. I untangle how relationships between citizens and states shape the construction of medical risk.     

The Effect of Exogenous β-<Emphasis Type="Italic">N-</Emphasis>Methylamino-<Emphasis Type="SmallCaps">l</Emphasis>-alanine on the Growth of <Emphasis Type="Italic">Synechocystis</Emphasis> PCC6803

β-N-Methylamino-l-alanine (BMAA), a non-proteinogenic amino acid, has been detected in a range of cyanobacteria, including terrestrial, aquatic, free living and endosymbiotic species. The widespread occurrence of cyanobacteria in the environment raises concerns regarding the ecological and toxicological impact of BMAA, and consequently, studies have focussed extensively on the toxicity and environmental impact of BMAA, while no research has addressed the ecophysiological or metabolic role of the compound in cyanobacteria. In this study, both the uptake of exogenous BMAA by and the effect of exogenous BMAA on the growth of Synechocystis PCC6803 were investigated. BMAA was rapidly taken up by the non-diazotrophic cyanobacterium Synechocystis PCC6803 in a concentration dependent manner. The presence of exogenous BMAA resulted in a substantial and concentration-dependent decrease in cell growth and the substantial loss of photosynthetic pigmentation. Similar effects were seen in the presence of the non-proteinogenic amino acid, 2,4-diaminobutyric acid but to a lesser degree than that of BMAA. The effects were reversed when light was decreased from 16 to 10 μmol m⁻² s⁻¹. Control cultures grown in the presence of l-arginine, l-asparagine, l-glutamate and glycine showed normal or slightly increased growth with no change in pigmentation. The decrease in growth rate coupled to bleaching indicates that BMAA may induce chlorosis in the presence of adequate photosynthetic radiation suggesting a connection between BMAA and the induction of conditions, such as nitrogen or sulphur depletion, that result in growth arrest and the induction of chlorosis.     

Yeast adaptor protein, Nbp2p, is conserved regulator of fungal Ptc1p phosphatases and is involved in multiple signaling pathways

Nbp2p is an Src homology 3 (SH3) domain-containing yeast protein that is involved in a variety of cellular processes. This small adaptor protein binds to a number of different proteins through its SH3 domain, and a region N-terminal to the SH3 domain binds to the protein phosphatase, Ptc1p. Despite its involvement in a large number of physical and genetic interactions, the only well characterized function of Nbp2p is to recruit Ptc1p to the high osmolarity glycerol pathway, which results in down-regulation of this pathway. In this study, we have discovered that Nbp2p orthologues exist in all Ascomycete and Basidiomycete fungal genomes and that all possess an SH3 domain and a conserved novel Ptc1p binding motif. The ubiquitous occurrence of these two features, which we have shown are both critical for Nbp2p function in Saccharomyces cerevisiae, implies that a conserved role of Nbp2p in all of these fungal species is the targeting of Ptc1p to proteins recognized by the SH3 domain. We also show that in a manner analogous to its role in the high osmolarity glycerol pathway, Nbp2p functions in the down-regulation of the cell wall integrity pathway through SH3 domain-mediated interaction with Bck1p, a component kinase of this pathway. Based on functional studies on the Schizosaccharomyces pombe and Neurospora crassa Nbp2p orthologues and the high conservation of the Nbp2p binding site in Bck1p orthologues, this function of Nbp2p appears to be conserved across Ascomycetes. Our results also clearly imply a function for the Nbp2p-Ptc1p complex other cellular processes.     

Predation has a greater impact in less productive environments: variation in roe deer, Capreolus capreolus, population density across Europe

Aim We aimed to describe the large‐scale patterns in population density of roe deer Caprelous capreolus in Europe and to determine the factors shaping variation in their abundance. Location Europe. Methods We collated data on roe deer population density from 72 localities spanning 25° latitude and 48° longitude and analysed them in relation to a range of environmental factors: vegetation productivity (approximated by the fraction of photosynthetically active radiation) and forest cover as proxies for food supply, winter severity, summer drought and presence or absence of large predators (wolf, Canis lupus, and Eurasian lynx, Lynx lynx), hunter harvest and a competitor (red deer, Cervus elaphus). Results Roe deer abundance increased with the overall productivity of vegetation cover and with lower forest cover (sparser forest cover means that a higher proportion of overall plant productivity is allocated to ground vegetation and thus is available to roe deer). The effect of large predators was relatively weak in highly productive environments and in regions with mild climate, but increased markedly in regions with low vegetation productivity and harsh winters. Other potentially limiting factors (hunting, summer drought and competition with red deer) had no significant impact on roe deer abundance. Main conclusions The analyses revealed the combined effect of bottom‐up and top‐down control on roe deer: on a biogeographical scale, population abundance of roe deer has been shaped by food‐related factors and large predators, with additive effects of the two species of predators. The results have implications for management of roe deer populations in Europe. First, an increase in roe deer abundance can be expected as environmental productivity increases due to climate change. Secondly, recovery plans for large carnivores should take environmental productivity and winter severity into account when predicting their impact on prey.     

Intrahepatic bile ducts transport water in response to absorbed glucose

The physiological relevance of theabsorption of glucose from bile by cholangiocytes remains unclear. Theaim of this study was to test the hypothesis that absorbed glucosedrives aquaporin (AQP)-mediated water transport by biliary epitheliaand is thus involved in ductal bile formation. Glucose absorption andwater transport by biliary epithelia were studied in vitro bymicroperfusing intrahepatic bile duct units (IBDUs) isolated from ratliver. In a separate set of in vivo experiments, bile flow andabsorption of biliary glucose were measured after intraportal infusionof D-glucose or phlorizin. IBDUs absorbedD-glucose in a dose- and phlorizin-dependent manner with anabsorption maximum of 92.8 ± 6.2 pmol · min¹ · mm¹.Absorption of D-glucose by microperfused IBDUs resulted inan increase of water absorption (J_v = 310nl · min¹ · mm¹,P_f = 40 × 10³ cm/sec).Glucose-driven water absorption by IBDUs was inhibited byHgCl₂, suggesting that water passively followsabsorbed D-glucose mainly transcellularly viamercury-sensitive AQPs. In vivo studies showed that as the amount ofabsorbed biliary glucose increased after intraportal infusion ofD-glucose, bile flow decreased. In contrast, as theabsorption of biliary glucose decreased after phlorizin, bile flowincreased. Results support the hypothesis that the physiologicalsignificance of the absorption of biliary glucose by cholangiocytes islikely related to regulation of ductal bile formation.

     

Channel-mediated water movement across enclosed or perfused mouse intrahepatic bile duct units

We previously reported the developmentof reproducible techniques for isolating and perfusing intactintrahepatic bile duct units (IBDUs) from rats. Given the advantages oftransgenic and knockout mice for exploring ductal bile formation, wereport here the adaptation of those techniques to mice and theirinitial application to the study of water transport across mouseintrahepatic biliary epithelia. IBDUs were isolated from livers ofnormal mice by microdissection combined with enzymatic digestion. Afterculture, isolated IBDUs sealed to form intact, polarized compartments,and a microperfusion system employing those isolated IBDUs developed. Aquantitative image analysis technique was used to observe a rapidincrease of luminal area when sealed IBDUs were exposed to a series of inward osmotic gradients reflecting net water secretion; the choleretic agonists secretin and forskolin also induced water secretion into IBDUs. The increase of IBDU luminal area induced by inward osmotic gradients and choleretic agonists was reversibly inhibited by HgCl₂, a water channel inhibitor. With the use of aquantitative epifluorescence technique in perfused mouse IBDUs, a highosmotic water permeability (P_f = 2.5-5.6 × 10² cm/s) was found in response toosmotic gradients, further supporting the presence of water channels.These findings suggest that, as in the rat, water transport acrossintrahepatic biliary epithelia in mice is water channel mediated.